Translocation of the Retinoblastoma Gene Product during Mitosis

A comparison between the two methods was performed for tyrosine phosphorylation of proteins in murine thymocytes exposed to low dose radiation using antiphosphotyrosine McAb.It was shown that tyrosine phosphorylation of 17.5 kD,22 kD,28 kD,32 kD,40 kD,43 kD and 53 kD protein molecules labelled metabolically with H332PO4 appeared in the thymocytes after radiation by immunoprecipitation technique and tyrosine phosphorylation of 28 kD,32 kD protein molecules appeared by western blotting.The results suggest that immunoprecipitation is superior to Western blotting in detection of protein tyrosine phosphorylation.

Immunoprecipitation

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Identification of interaction between ISCA1 and NLS-RARα by co-immunoprecipitation in mammalian cells

Di-Si Junyi Daxue xuebao (2009)

Dong Wang

AIM:To verify the interaction between ISCA1 and NLS-RARα by co-immunoprecipitation and Western blotting.METHODS:The eukaryotic expression vector pCMV-HA-NLS-RARα and pCMV-Myc-ISCA1 were constructed,identified and then transfected into human embryo kidney 293 cells.Co-immunoprecipitation and Western blotting were used to investigate the interaction between ISCA1 and NLS-RARα.RESULTS:After being verified,eukaryotic expression vectors were co-transfected into HEK 293 cells,HA-NLS-RARα protein was then immunoprecipitated by anti-HA polyclonal antibody,and Myc-ISCA1 protein was detected by western blotting with anti-Myc monoclonal antibody from the immunoprecipitared complex.CONCLUSION:Eukaryotic expression vectors with HA-NLS-RARα and Myc-ISCA1 are constructed successfully.The interaction between ISCA1 and NLS-RARα is identified by co-immunoprecipitation and Western blotting in vitro.

Immunoprecipitation

HEK 293 cells

NLS

Polyclonal antibodies

Chromatin immunoprecipitation

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Analysis of Signalling Pathways by Western Blotting and Immunoprecipitation

Methods in molecular biology (2011)

Aymen I. Idris

Immunoprecipitation

10.1007/978-1-61779-415-5_15

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Immunoprecipitation: Western Blot for Proteins of Low Abundance

Methods in molecular biology (2015)

Edward P. Trieu Ira N. Targoff

Immunoprecipitation

10.1007/978-1-4939-2694-7_34

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SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting

Methods in molecular biology (2012)

Edward P. Trieu Ira N. Targoff

Immunoprecipitation

Sodium dodecyl sulfate

10.1007/978-1-61779-821-4_18

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Immunoprecipitation and Blotting

Springer protocols handbooks/Springer protocols (2009)

Stephen Thompson

Immunoprecipitation and blotting both use antibodies (normally, but not exclusively monoclonal antibodies) to detect and quantitate specific protein antigens in complex cellular or protein mixtures. Immunoprecipitation has an advantage in that the antigens are allowed to react with the antibodies in their native conformation prior to their subsequent separation and quantification. A further advantage is that a protein at a very low concentration can be concentrated from the relatively large volume of 1–2 mL. The major disadvantage is that the proteins normally have to be radiolabeled to facilitate their detection. In Western blotting the proteins do not have to be labeled, but they have to be separated by electrophoresis in polyacrylamide gels prior to their transfer to either nitrocellulose, polyvinyldifluoride (PVDF), or nylon membranes. This seriously restricts the size of the sample, and hence the protein antigen has to be present at higher concentrations. A further disadvantage is that the antigen is not normally in its native conformation when it reacts with the antibody, because the electrophoresis usually being carried out in the presence of sodium dodecyl sulfate (SDS) to maximize the resolution of the separated proteins. If an antibody has a lower affinity for an antigen, it may well immunoprecipitate an antigen but not react with it on a Western blot. This is the reason why some workers slot-blot their protein mixtures rather than separate them by electrophoresis. This maintains their native conformation. The main problem encountered here is that crossreactions of the primary antibody with all the other proteins in the mixture can outweigh the antigen-specific binding. An unlabeled antigen is often therefore prepurified using an immobilized lectin or even immunoprecipitation itself prior to its quantitation on a slot-blot. Such a combination of lectin and antibody techniques has tremendous potential both in more precise analysis of cellular antigens and in the characterization of disease progression.

Immunoprecipitation

Sodium dodecyl sulfate

10.1007/978-1-59745-198-7_196

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